Volume 14 Number 7 July 2012 pp. 559–571 559 www.neoplasia.com † Hiroshi Katoh*, , Keishi Yamashita*, Mina Waraya*, Epigenetic Silencing of HOPX †,‡ § Ofer Margalit , Akira Ooki*, Hideaki Tamaki , Promotes Cancer Progression Hiroyuki Sakagami§, Kenichi Kokubo¶, # in Colorectal Cancer1,2,3 David Sidransky and Masahiko Watanabe* *Department of Surgery, Kitasato University School of Medicine, Sagamihara, Japan; †Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX; ‡Talpiot Medical Leadership Program, Chaim Sheba Medical Center, Tel-Hashomer, Israel; §Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Japan; ¶Department of Medical Engineering and Technology, School of Allied Health Sciences, Kitasato University, Sagamihara, Japan; #Head and Neck Cancer Research Division, Department of Otolaryngology, The Johns Hopkins University School of Medicine, Baltimore, MD Abstract Homeodomain-only protein X (HOPX)-β promoter methylation was recently shown to be frequent in human cancers and was suggested as tumor suppressor gene in esophageal and gastric cancer. The aim of this study was to inves- tigate the mechanistic roles of HOPX-β promoter methylation and its clinical relevance in colorectal cancer (CRC). HOPX-β promoter methylation was assessed in human CRC cell lines and 294 CRC tissues. HOPX mRNA and pro- tein levels were measured in relation to HOPX-β promoter methylation. The effects of forced HOPX expression on tumorigenesis were studied using in vitro and in vivo assays. The association between HOPX-β promoter methylation and clinical relevance of CRC patients was determined. HOPX-β promoter methylation is cancer-specific and fre- quently found in CRC cell lines and tissues, resulting in the down-regulation of HOPX mRNA and protein levels. In CRC cell lines, forced expression of HOPX suppressed proliferation, invasion, and anchorage-independent growth. DNA microarray analyses suggested critical downstream genes that are associated with cancer cell proliferation, in- vasion or angiogenesis. In a mouse xenograft model, HOPX inhibited tumorigenesis and angiogenesis. Finally, HOPX-β promoter methylation was associated with worse prognosis of stage III CRC patients (hazard ratio = 1.40, P = .035) and also with poor differentiation (P = .014). In conclusion, HOPX-β promoter methylation is a frequent and cancer-specific event in CRC progression. This epigenetic alteration may have clinical ramifications in the diagnosis and treatment of CRC patients. Neoplasia (2012) 14, 559–571 Address all correspondence to: Hiroshi Katoh, MD, PhD, or Masahiko Watanabe, MD, PhD, FACS, Kitasato University School of Medicine, Kitasato 1-15-1, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. E-mail: [email protected] 1The authors have no current external funding sources for this study. The authors declare no conflict of interest. 2This article refers to supplementary materials, which are designated by Tables W1 to W6 and Figures W1 and W2 and are available online at www.neoplasia.com. 3Our study showed first that HOPX promoter hypermethylation is cancer-specific and frequent in human colorectal cancer. HOPX was found to have multifunctional tumor suppressive activity and suppressed tumorigenesis and angiogenesis in vivo. HOPX promoter hypermethylation independently shows worse prognosis in patients with stage III disease. Importantly, patients with N1-stage III disease without HOPX promoter hypermethylation demonstrated excellent prognosis, comparable to that of stage II patients, suggesting reconsideration of the adjuvant chemotherapy currently indicated in these patients with stage III disease. Received 9 February 2012; Revised 12 June 2012; Accepted 14 June 2012 Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00 DOI 10.1593/neo.12330 560 HOPX in Colorectal Cancer Katoh et al. Neoplasia Vol. 14, No. 7, 2012 Introduction in each). Mice were injected with 1 × 106 cells on the left flank Colorectal cancer (CRC) is the second most prevalent cancer and the with either HCT116 or DLD1 cells transfected with a mock vector fourth leading cause of cancer death worldwide [1]. Although CRC as control; and on the right flank, with cells transfected with a screening, that is, fecal occult blood test and colonoscopy, has con- HOPX-expressing vector. Tumor volumes were calculated from caliper tributed to a reduction in mortality [2], 70% of newly discovered measurements of two orthogonal diameters (larger (x) and smaller CRC patients are still detected at an advanced stage, resulting in poor (y) diameters) by using the formula: volume = xy2/2. All animal experi- prognosis [3]. Recently, sophisticated scientific technologies have ments were performed in strict accordance with the guidelines for emerged, such as DNA microarray, pharmacological unmasking epi- animal experiments of the Kitasato University School of Medicine. genetic profiles, and proteomic techniques, advancing the discovery of novel biomarkers for CRC [4]. Human CRC Tissues Alterations in DNA methylation, an epigenetic process present in Paraffin-embedded primary CRC tissues and paired normal mucosa mammalian cells, are one of the hallmarks of cancer [5]. The promoter specimens were obtained from 99 patients diagnosed with stage I to regions of many genes, particularly housekeeping genes, are populated by IV CRC (Table W1), and 146 patients diagnosed with stage III CRC CpG dinucleotides, which are often underrepresented in the remainder (Table W2), used for the quantitative methylation-specific polymerase of the genome. These regions have been termed “CpG islands” because chain reaction (Q-MSP). An independent set of 22 samples was sub- they are protected from methylation in normal cells [6]. This protection is jected to immunohistochemistry assays. Snap-frozen tissues were taken critical because methylation of CpG islands is associated with loss of ex- from 27 patients diagnosed with stage I-IV CRC, used for the Q-MSP pression of that particular gene. In carcinogenesis, global hypomethyla- assay coupled with reverse transcription–polymerase chain reaction tion [6,7] is often accompanied by dense hypermethylation of specific (RT-PCR). All specimens were taken during either potentially curative promoters [5]. Many studies have demonstrated that silencing of tumor resection (stages I-III patients), or palliative resection (stage IV pa- suppressor genes (TSGs) is associated with promoter hypermethylation tients). TNM classification was made according to the UICC (Unio in human cancers and serves as an alternative mechanism for loss of func- Internationalis Contra Cancrum) staging system. All individuals gave tion. However, cancer-specific methylation is rather a rare event and is written informed consent for pathologic assessment and molecular test- usually evident only in genes with a robust tumor suppressive function. ing on their samples. This study was performed with approval of the We previously developed pharmacologic reversal of epigenetic silenc- Ethics Committee of the Kitasato University School of Medicine. ing and uncovered a myriad of transcriptionally repressed genes in human cancers [8]. Using this technique, we have identified several novel TSG Bisulfite Treatment of DNA and Sequencing Analysis candidates, such as PGP9.5 [9], NMDAR2A/B [10,11], and NEFH Tissue sections from tumor and corresponding normal mucosa were [12]. Among these candidates, homeodomain-only protein X (HOPX) stained with hematoxylin and eosin and dissected under microscope. was of particular interest because its methylation pattern was found Genomic DNA was subsequently extracted using QIAamp DNA FFPE to be cancer-specific [8]. Indeed, HOPX-β methylation was correlated Kit (Qiagen Sciences, Hilden, Germany). Genomic DNA from cell with tumorigenesis and worse prognosis in gastric and esophageal can- lines was extracted using QIAamp DNA Mini Kit (Qiagen). Bisulfite cers [13,14]. In the present study, we investigated the importance of treatment was carried out with EpiTect Bisulfite Kit (Qiagen) and was HOPX-β promoter methylation in CRC. We assessed the methylation subsequently amplified by PCR. Primer sequences for the HOPX-β status of HOPX-β promoter in CRC cell lines and tissues and looked promoter region were designed to recognize DNA alterations caused for its functional role and clinical relevance. by bisulfite treatment (Table W3). The PCR products were either di- rectly sequenced (Figure 1B) or purified with QIAquick Spin (Qiagen) Materials and Methods and inserted into a pCR4-TOPO vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA; Figure 1D). Cell Lines CRC cell lines (HCT15, DLD1, COLO205, and LoVo) and an Quantitative Methylation-Specific PCR esophageal squamous cell carcinoma cell line (TE15) were kindly pro- For quantitative methylation analysis, TaqMan Q-MSP was carried vided by the Cell Response Center for Biomedical Research Institute out using iQ SuperMix (Bio-Rad Laboratories, Hercules, CA) as of Development, Aging and Cancer, Tohoku University (Sendai, reported [11]. All reactions were performed in triplicates. Primer Japan). HCT116 and SW480 were purchased from American Type sequences and PCR conditions are described in Table W3. The most Culture Collection (Manassas, VA). Other CRC cell lines (COLO320, frequent location of restriction CpG sites for translation has been CW2, and CACO2) were obtained from RIKEN BioResource reported to be around 500 bp upstream of translation